A core objective of tissue biology is to understand how cells interact to generate tissues and organs. While great strides have been made in defining signaling pathways that mediate cellular interactions, there is a fundamental gap in our knowledge concerning how these signaling systems are integrated during mammalian organogenesis. Bridging this knowledge gap is important in identifying druggable targets for cancer, as tumor growth and progression is marked by disorganization of tissue structure, and deregulation of specialized cell populations, such as stem cells. The goal of this application is to understand how extracellular factors, SLITs, influence tissue morphogenesis by regulating the proliferation of a single layer of basal cells in which stem cells reside. Here, mammary branching morphogenesis is used as a model system and, guided by strong preliminary data, the following model is investigated in which a key inhibitor of mammary branching, TGF-21, restrains basal cell proliferation and adhesion by upregulating signaling through the SLIT/ROBO1 pathway. This, in turn, controls basal cell growth and branching morphogenesis by increasing the adhesive functions of 2-catenin at the membrane, at the expense of its proliferative functions in the nucleus. A variety of cell biological, imaging and biochemical techniques will be employed, as well as powerful transplant techniques to manipulate genetically modified mammary tissue. Three hypotheses are tested in three Aims: I) that TGF-21 upregulates Robo1 expression, specifically in basal cells, and that this inhibits branching by restraining basal cell growth;II) that SLIT/ROBO1 signaling opposes the actions of canonical WNTs by altering the subcellular localization of 2-catenin and promoting its cell adhesive functions at the expense of its transcriptional functions;and III), that SLITs are non-renewal factors for stem cells that function to counter the self-renewal signals of canonical WNT signaling by regulating 2-catenin. Together, these experiments will have a positive impact on cancer biology of breast and other glandular organs containing basal stem cell subpopulations (e.g. prostate, salivary etc), because they delineate a novel tumor suppressive signaling network that appears to function in stem cells. PUBLIC HEALTH RELEVANCE: Breast cancer is the second leading cause of cancer deaths in women. Tumor progression from ductal carcinoma in situ to infiltrating ductal carcinoma requires disruption of the outer, basal layer of breast cells that also contains breast stem cells. This study investigates a novel tumor suppressive network of signaling pathways that restricts the growth and maintains integrity of this critical, gate keeping layer. These studies have the potential to identify therapeutically-relevant targets in metastasis.